Strategic Single-Residue Substitution in the Antimicrobial Peptide Esc(1-21) Confers Activity against Staphylococcus aureus , Including Drug-Resistant and Biofilm Phenotype.

Staphylococcus aureus , a bacterium resistant to multiple drugs, is a significant cause of illness and death worldwide. Antimicrobial peptides (AMPs) provide an excellent potential strategy to cope with this threat. Recently, we characterized a derivative of the frog-skin AMP esculentin-1a, Esc(1-21) ( 1 ) that is endowed with potent activity against Gram-negative bacteria but poor efficacy against Gram-positive strains. In this study, three analogues of peptide 1 were designed by replacing Gly 8 with α-aminoisobutyric acid (Aib), Pro, and dPro ( 2 - 4 , respectively). The single substitution Gly 8 → Aib 8 in peptide 2 makes it active against the planktonic form of Gram-positive bacterial strains, especially Staphylococcus aureus , including multidrug-resistant clinical isolates, with an improved biostability without resulting in cytotoxicity to mammalian cells. Moreover, peptide 2 showed a higher antibiofilm activity than peptide 1 against both reference and clinical isolates of S . aureus . Peptide 2 was also able to induce rapid bacterial killing, suggesting a membrane-perturbing mechanism of action. Structural analysis of the most active peptide 2 evidenced that the improved biological activity of peptide 2 is the consequence of a combination of higher biostability, higher α helical content, and ability to reduce membrane fluidity and to adopt a distorted helix, bent in correspondence of Aib 8 . Overall, this study has shown how a strategic single amino acid substitution is sufficient to enlarge the spectrum of activity of the original peptide 1 , and improve its biological properties for therapeutic purposes, thus paving the way to optimize AMPs for the development of new broad-spectrum anti-infective agents.